Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay

Abstract

Summary Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.

Linear models enable powerful differential activity analysis in massively parallel reporter assays

“…For the allelic studies, we use a random effects model for mpralm and paired t-tests. Both Tewhey et al (2016) and Ulirsch et al (2016) compare alleles in different cellular contexts; we observe similar behavior of all evaluations in all contexts (data not shown) and have therefore chosen to depict results from one cellular context for both of these studies. For Tewhey et al (2016) we depict results both from a large pool of elements used for initial screening and a smaller, targeted pool.…”

“…Studies comparing different alleles (Tewhey et al, 2016;Ulirsch et al, 2016), are naturally paired in the sense that both alleles are measured at the same time in the same sample. We can model that using mpralm by using a random effect representing the loci.…”

“…Versions can correspond to allelic versions of a sequence (Ulirsch et al, 2016;Tewhey et al, 2016;Vockley et al, 2015) or different environmental contexts (Inoue et al, 2017), such as different cell or tissue types . These studies have compared different sequences versions using paired t-tests, rank sum tests, and Fisher's exact test (by pooling counts over biological replicates).…”

Variant Interpretation: Functional Assays to the Rescue

“…13,[55][56][57] For example, MPRAs have been used to quantify the effects of more than 100,000 variants of three liver enhancers. 58 MPRAs have also been used to simultaneously test thousands of variants associated with eQTLs 22 or variants in linkage disequilibrium with lead SNPs from GWASs for red blood cell traits. 59 Another noteworthy adaptation of MPRAs is population-scale self-transcribing active regulatory region sequencing (POPSTARR), in which candidate regulatory elements from numerous individuals are cloned via DNA sequence capture and tested in parallel.…”

“…20,21 Assessment of tens of thousands of eQTLadjacent, and therefore possibly functional, transcriptional regulatory variants revealed a few hundred that influenced expression. 22 These examples illustrate how the rich and comprehensive datasets that MAVEs produce can generate predictions that are much more accurate than those of currently available variant-effect-prediction algorithms.…”